Vaginal Tissue Biopsies Research Articles

Induction of Cellular Senescence in Rat Vaginal Fibroblasts and Treatment With Senolytics: An in Vitro Model for the Study of Pelvic Organ Prolapse.

The objective of this study was to develop an in vitro model of cellular senescence using rat vaginal fibroblasts and determine the effects of treatment with senolytics. Rat vaginal tissue biopsies were collected. Primary vaginal fibroblasts were isolated and characterized by immunofluorescence. To induce cellular senescence, fibroblasts were treated with etoposide at 3, 10, and 20 mM for 24 hours, followed by treatment with the senolytics dasatinib (1 mM) and/or quercetin (20 mM). After treatment, RNA was extracted and the expression of selected genes was quantified. Immunostaining of senescence markers was also performed. Fibroblasts were confirmed by positive immunostaining for α-smooth muscle actin and vimentin, and negative immunostaining for pan-cytokeratin. Treatment with etoposide resulted in a dose-dependent increase in expression of the senescence-associated secretory phenotype markers MMP-7, MMP-9, and IL-b1 (P < 0.05) compared with controls. Immunostaining showed increased expression of γ-H2A and p21 after treatment with etoposide. Cells treated with dasatinib and quercetin after etoposide treatment had decreased expression of p21, MMP-7, MMP-9, and IL-1b compared with cells treated only with etoposide (P < 0.05). Upregulation of senescence-associated factors provided evidence that senescence can be induced in vaginal fibroblasts in vitro. Furthermore, treatment with the senolytics dasatinib and quercetin abrogated the senescence phenotype induced by etoposide in rat vaginal fibroblasts. Our findings provide a novel model for the study and development of new therapies targeting the disordered extracellular matrix associated with pelvic organ prolapse.

Vulvovaginal candidiasis: histologic lesions are primarily polymicrobial and invasive and do not contain biofilms

The recent demonstration of a vaginal biofilm in bacterial vaginosis and its postulated importance in the pathogenesis of recurrent bacterial vaginosis, including relative resistance to therapy, has led to the hypothesis that biofilms are crucial for the development of vulvovaginal candidiasis. The histopathology and microbial architecture of vulvovaginal candidiasis have not been previously defined; neither has Candida, containing biofilm been reported in situ. The present study aimed at clarifying the histopathology of vulvovaginal candidiasis including the presence or absence of vaginal biofilm. In a cross-sectional study, vaginal tissue biopsies were obtained from 35 women with clinically, microscopically, and culture-proven vulvovaginal candidiasis and compared with specimens obtained from 25 healthy women and 30 women with active bacterial vaginosis. Vaginal Candida infection was visualized using fluorescent in situ hybridization with ribosomal gene-based probes. Candida microorganisms were confirmed in 26 of 35 biopsies obtained from women with vulvovaginal candidiasis; however, Candida containing biofilm were not detected in any of the cases. Histopathological lesions were exclusively invasive and accompanied by co-invasion with Gardnerella or Lactobacillus species organisms. Histopathological lesions of vulvovaginal candidiasis are primarily invasive in nature and polymicrobial and do not resemble biofilms. The clinical significance of Candida tissue invasion is unknown.

Static Mechanical Loading Influences the Expression of Extracellular Matrix and Cell Adhesion Proteins in Vaginal Cells Derived From Premenopausal Women With Severe Pelvic Organ Prolapse.

Primary human vaginal cells derived from women with severe pelvic organ prolapse (POP-HVCs) demonstrate altered cellular characteristics as compared to cells derived from asymptomatic women (control-HVCs). Using computer-controllable Flexcell stretch unit, we examined whether POP-HVCs react differently to mechanical loading as compared to control-HVCs by the expression of extracellular matrix (ECM) components, cell-ECM adhesion proteins, and ECM degrading and maturating enzymes. Vaginal tissue biopsies from premenopausal patients with Pelvic Organ Prolapse Quantification System stage ≥3 (n = 8) and asymptomatic controls (n = 7) were collected during vaginal hysterectomy or repair. Human vaginal cells were isolated by enzymatic digestion, seeded on collagen (COLI)-coated plates, and stretched (24 hours, 25% elongation). Total RNA was extracted, and 84 genes were screened using Human ECM and Adhesion Molecules polymerase chain reaction array; selected genes were verified by quantitative reverse transcription-polymerase chain reaction. Stretch-conditioned media (SCM) were collected and analyzed by protein array, immunoblotting, and zymography. In mechanically stretched control-HVCs, transcript levels of integrins (ITGA1, ITGA4, ITGAV, and ITGB1) and matrix metalloproteinases (MMPs) 2, 8, and 13 were downregulated (P < .05); in POP-HVCs, MMP1, MMP3, and MMP10, ADAMTS8 and 13, tissue inhibitor of metalloproteinases (TIMPs) 1 to 3, ITGA2, ITGA4, ITGA6, ITGB1, contactin (CNTN1), catenins (A1 and B1), and laminins (A3 and C1) were significantly upregulated, whereas COLs (1, 4, 5, 6, 11, and 12) and LOXL1 were downregulated. Human vaginal cells massively secrete MMPs and TIMPs proteins; MMP1, MMP8, MMP9 protein expression and MMP2 gelatinase activity were increased, whereas TIMP2 decreased in SCM from POP-HVCs compared to control-HVCs. Primary human vaginal cells derived from women with severe pelvic organ prolapse and control-HVCs react differentially to in vitro mechanical stretch. Risk factors that induce stretch may alter ECM composition and cell-ECM interaction in pelvic floor tissue leading to the abatement of pelvic organ support and subsequent POP development.

Comparative Characterization of Vaginal Cells Derived From Premenopausal Women With and Without Severe Pelvic Organ Prolapse.

This study tested a hypothesis that primary human vaginal cells derived from tissue of premenopausal women with severe pelvic organ prolapse (POP-HVCs) would display differential functional characteristics as compared to vaginal cells derived from asymptomatic women with normal pelvic floor support (control-HVCs). Vaginal tissue biopsies were collected from premenopausal patients with POP (n = 8) and asymptomatic controls (n = 7) during vaginal hysterectomy or repair. Primary vaginal cells were isolated by enzymatic digestion and characterized by immunocytochemistry. Cell attachment and proliferation on different matrices (collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin, and vitronectin) were compared between POP-HVCs and control-HVCs. RNA was extracted, and the expression of 84 genes was screened using Human Extracellular Matrix and Adhesion Molecules RT(2) Profiler PCR array. The expression of selected genes was verified by quantitative reverse transcription-polymerase chain reaction. (1) Control-HVCs attached to collagen IV more efficiently than POP-HVCs; (2) control-HVCs and POP-HVCs show a similar proliferation rate when plated on proNectin and collagen I; (3) when seeded on collagen I, resting POP-HVCs expressed significantly (P < .05) increased transcript levels of collagen VII, multiple matrix metalloproteinases (MMP3, MMP7, MMP10, MMP12, MMP13, and MMP14), integrins (ITGA1, ITGA4, ITGA6, ITGA8, ITGB1, ITGB2, and ITGB3), and cell adhesion molecules as compared to control-HVCs. Collagen XV and tissue inhibitors of MMPs (TIMP1 and TIMP2) as well as genes involved in the biogenesis and maturation of collagen and elastin fibers (LOX, LOXL1-LOXL3, BMP1, and ADAMTS2) were significantly downregulated in POP-HVCs versus control-HVCs (P < .05). Resting primary POP-HVCs in vitro show altered cellular characteristics as compared to control-HVCs, which may influence their dynamic responses to external mechanical or hormonal stimuli.

Single and Multiple Dose Pharmacokinetics of Dolutegravir in the Genital Tract of HIV-Negative Women

Antiretrovirals that achieve adequate concentrations in anatomical sites of transmission are of interest for HIV prevention. A Phase I open-label pharmacokinetic (PK) study was performed to describe first dose (PK1) and steady-state (PK2) PKs of the integrase inhibitor dolutegravir (DTG) in blood plasma (BP), cervicovaginal fluid (CVF), cervical tissue (CT) and vaginal tissue (VT) in HIV type-1-negative women. A total of 8 healthy females given DTG 50 mg daily for 5-7 days had 11 paired BP and CVF samples collected over 24 h following the first dose (PK1) and multiple dosing (PK2). Each woman underwent CT and VT biopsies at 1/4 time points at PK1 and PK2 to generate composite PK profiles. DTG concentrations were analysed by validated liquid chromatography-tandem mass spectrometry methods. Non-compartmental PK analysis was performed and Spearman rank correlations determined between matrices. BP areas under the concentration-time curve (AUCs) were similar to previous reports and concentrations remained greater than the protein-adjusted (PA) 90% inhibitory concentration (IC90) for wild-type HIV (64 ng/ml). CVF exposures were approximately 6% of BP with low inter-individual variability. CT and VT exposures were 7% of BP at PK1, and 9-10% of BP at PK2 with 94% of samples >PA-IC90. CT and VT concentrations were correlated to each other (ρ=0.70, P=0.003), and to CVF at steady state (ρ=0.52, P=0.04). Accumulation of DTG from PK1 to PK2 occurred in BP, CT and VT, but only marginally in CVF. DTG BP PK were consistent with previously published values. CVF, CT and VT exposures were highly correlated. At PK2, DTG accumulated to a greater extent in tissue than in BP or CVF, suggesting increased tissue affinity.

Multi-Compartmental Transport of Anti-HIV Molecules into and through Mucosa

Sexual transmission of HIV derives from contact of virions with host cells, e.g. in the epithelium and lamina propria of the vagina. Current antiviral drugs (e.g. the reverse transcriptase inhibitor Tenofovir) show promise for topical mucosal prophylactic action against transmission (termed “microbicides”). They can be introduced by multiple delivery systems, e.g. gels and intravaginal rings. Their success (pharmacodynamics) depends upon adequate distribution in time and space (pharmacokinetics) vs. distribution of invading virions. We have created a family of mechanistic transport models of microbicide molecules, as they distribute from different vehicles into and through vaginal fluids and mucosal tissues, and interact with host cells and virions. We report here two new models that characterize transport of antiviral molecules from a vaginal gel or ring into and through the vaginal epithelium and lamina propria, with uptake within the latter into blood capillaries. The models contain systems of coupled convection-diffusion equations - one for each compartment. These are solved numerically (MatLab and Comsol for gels and rings, respectively). We have applied these models to transport of Tenofovir, and compared results with in vivo measurements of drug levels (vaginal fluid, tissue, bloodstream) in two human studies of a 1% Tenofovir gel (CONRAD; MTN-001). The models show good agreement in predicting the drop in Tenofovir concentrations (vaginal fluids; biopsy) during 24h after a single application of 4 mL of gel (CONRAD), and also the quasi-steady state values in vaginal tissue biopsies, obtained during continuous once-per-day application of 4 mL of gel (MTN-001). We illustrate use of the models to understand tradeoffs amongst the packaging of drug in delivery vehicles with different properties and volumes/geometries, in relation to the frequency of application needed to create and sustain prophylactic drug levels in target mucosal compartments (NIH U19 AI077289).

Maraviroc Concentrates in the Cervicovaginal Fluid and Vaginal Tissue of HIV-Negative Women

To compare single- and multiple-dose maraviroc exposures in cervicovaginal fluid (CVF) and vaginal tissue (VT) with blood plasma (BP) and quantify maraviroc protein binding in CVF. Open-label pharmacokinetic study. In 12 HIV-negative women, 7 paired CVF and BP samples were collected over 12 hours after 1 maraviroc dose. Subjects then received maraviroc twice daily for 7 days. After the last dose, subjects underwent CVF and BP sampling as on day 1, with additional sampling during terminal elimination. VT biopsies were obtained at steady state. Day 1 and day 7 median maraviroc CVF AUCtau were 1.9- and 2.7-fold higher, respectively, than BP. On day 1, 6 of 12 subjects had detectable maraviroc CVF concentrations within 1 hour; 12 of 12 were detectable within 2 hours, and all exceeded the protein-free IC90. On day 7, maraviroc CVF protein binding was 7.6% and the VT AUCtau was 1.9-fold higher than BP. Maraviroc CVF concentrations 72 hours after dose and BP concentrations 12 hours after dose were similar. Higher maraviroc exposure in the female genital tract provides a pharmacologic basis for further evaluation of chemokine receptor 5 antagonists in HIV infection prophylaxis. This is the first study to report antiretroviral VT concentrations, CVF protein binding, and CVF terminal elimination.